VETERINARY DRUG RELEASE BY NANOSTRUCTURED POLYMERS

Juliano E. Oliveira1, Ed H. Madureira2, Luiz H.C. Mattoso3, Odílio B.G. Assis3

1Depto. de Engenharia de Materiais, Universidade Federal da Paraíba, João Pessoa (PB), Brasil

2Depto. de Reprodução Animal, Universidade de São Paulo, Pirassununga (SP), Brasil

3Lab. Nacional de Nanotecnologia para o Agronegócio, Embrapa Instrumentação, São Carlos (SP), Brasil

E-mail: juliano@ct.ufpb.br

Abstract. Several techniques are used to obtain polymeric nanofibers. Solution blow spinning (SBS) is a technology feasible to produce nanostructured polymeric membranes loaded with active agents. In the present study, nanofibrous mats of biopolymers like poly(lactic acid) (PLA) and zein loaded with medroxyprogesterone (MP4) were produced by SBS at different P4 concentrations. The spun membranes were characterized by scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). Interactions between progesterone and PLA were confirmed by rheological measurements of the biopolymer/P4 solutions and in the spun mats by microscopy (SEM), and thermal (DSC) analyses. The in vitro releasing of MP4 was evaluated using a high-performance liquid chromatography (HPLC). SEM micrographs provided evidences of a smooth and homogeneous structure for nanostructured membranes without progesterone crystals on fiber surface. Moreover, the presence of progesterone affects the fiber morphology. X-Ray analysis indicated that size of PLA crystallites increased with progesterone content. Finally, by in vitro release experiments it was possible to observe that the progesterone releasing in PLA fibers follows nearly first-order kinetics, probably due to the diffusion of hormone into solid matrix.

Keywords: biopolymer, hormone, drug release, veterinary medicine

1.INTRODUCTION

The use of nanofibers as carrier is promising for biomedical and agricultural applications[1, 2]. In controlled and sustained release applications, the active chemicals are dissolved in the polymer solution and incorporated into nanofibers by using a one-step method like electrospinning[3, 4]. Compared to the traditional bulk materials, these nanostructured membranes largely facilitated the drug delivery processes in many aspects of pharmacology[5]. The fibrous structure possesses the advantages of having a three-dimensional open porous medium with high surface area, what improves therapeutic efficacy and reduces toxicity[6, 7]. Due to the high surface area and the porous structure of these nanofibers applications in drugs delivery systems such as the liberation of anti-neoplastic[8], antibiotics[9], and pheromone agents have been suggested[6]. Other appealing advantages reported to nanofibers obtained by electrospinning are high encapsulation efficiency, high loading capacity, and cost-effectiveness[8]. Solution blow spinning (SBS) is an alternative method to obtain polymeric nanofibers[10, 11]. This technique produces fibers in the same size range as electrospinning, although with a greater potential for commercial scale-up[10]. Compared to the electrospinning, SBS is faster and operates at higher injection rates. Furthermore, SBS process does not use high voltage or electrically conductive collector to generate nanofibrous membranes[12, 13]. Like electrospinning, SBS of active agents in polymer solutions is expected to result in nanofibers with high surface area, fundamental to enhance the water solubility and the dissolution rate of chemicals[14]. Moreover, these can be designed to provide rapid, immediate, or delayed dissolution with sustained release behavior. The aforementioned features along with the ability to spin several polymers without altering the basic setup make the SBS fibers an attractive technology for application in delivery systems.

Biopolymers have been extensively used as material for drugs encapsulation due to their biocompatibility[15]. Among these polymers, poly(lactic acid) is widely used in medical applications such as drug delivery devices, absorbable sutures, and as materials for medical implants and other related applications[16-18]. Poly(lactic acid) has received great attention because its monomer, L-lactic acid, can be obtained by fermentation from renewable resources such as starches and sugars[16].

The release of hydrophobic active agents from electrospun PLA fibers is reported to follow a nearly zero-order kinetics due to the degradation mode of the fibers[14]. Moreover Zeng et al.[19] registered a burst release for hydrophilic active agents in PLA fibers via diffusion of the drug on or nearby the fiber’s surfaces.

Zein is considered a prolamine, the major storage protein of corn, which comprises about 45–50% of its protein content[20]. Isolated, zein is not used directly for human consumption due to its negative nitrogen balance and poor solubility in water. As a biopolymer which provides many advantages in such terms as biocompatibility, biodegradability, electrospinnability, and film forming ability, it is considered a potential raw material for bioengineering applications[21].

One interesting agent to be tested in such delivery system is the progesterone that consist in a progestin involved in their pregnancy[22]. In veterinary medicine, progesterone is used as a potent drug for suppression of estrous and ovulation, making possible the synchronization of the estrous and ovulation cycles in livestock animals[23]. Currently, the estrous control of livestock animals is conducted by the inserption of homogenous dispersions of progesterone embeds in silicone bands. However, the disadvantages of these inserts, according to Rathbone et al.[23] lie in the use of nondegradable polymers and the requirement for a disposal of these bands after their use. Thus, the association of progesterone and poly(lactic acid) matrix can be an excellent options for a bioabsorption material, such as for the manufacturing of intravaginal drug delivery systems[16].

In this sense, the present study aims to investigate the morphology characteristics of medroxyprogesterone encapsulated in poly(lactic acid) fibers obtained by solution blow spinning, and compared with those on zein electrospun fiber considering the effect of P4 addition on the morphology and structural fiber properties.

2.MATERIALS AND METHODS

2.1Materials

Poly(lactic acid) (PLA, Mn =75,000 g/mol) was obtained from Biomater Co. (Brazil). Zein from maize and progesterone (P4, 99%) from Sigma-Aldrich (USA). Chloroform, alcohol and acetone, used as solvent, were purchased from Synth (Brazil). Dialysis membranes (cut-off 12,000 Da) were purchased from Sigma-Aldrich (USA).

2.1Experimental

Preparation of Polymer Solutions

The solution formulations for SBS were prepared by weighting amounts of PLA, zein and P4 according to proportions listed in tables 1and 2. Dissolution was carried out in chloroform:acetone at 3:1 (v/v) for PLA and alcohol : water at 8:2 (v/v) for zein under vigorous stirring for several hours at room temperature, until complete dissolution.

Table 1 Poly(lactic acid) solution concentration of spun fibers.

PLA

Concentration (wt.%)

P4

Concentration (w/w %)

6

0

6

2

6

4

6

8

Table 2 Zein solution concentration of spun fibers.

Zein

Concentration (wt%)

P4

Concentration (w/w %)

20

0

20

2

20

4

20

8

Characterization of Solutions

For characterization of solutions the intrinsic viscosity of solution (η) was determined for all polymer concentrations at 25 oC in a range of 10-100 s-1 shear rate, using an Anton Paar Physica MCR rheometer. Concentric cylinders geometry (R = 24 and 28 mm diameters) were used. All measurements were performed in triplicate and values expressed as the mean.

Fiber Spinning

Zein fibers prepared by electrospinning were spun using a voltage of 24 kV, working distance of 12 cm, and a feed rate of 2 µL.min-1. Poly(lactic acid) fibers obtained by solution blow spinning were prepared under an air pressure of 0.4 MPa, working distance of 12 cm, and feed rate of 120 µL.min-1. Experimental operation details can be found elsewhere[10, 11]. In both cases, the generated nanofibers were collected on a rotating drum (200 rpm), wrapped with aluminum foil, generating nonwoven fiber mats. The fiber mats were than collected by peeling off and stored in a desiccator.

Characterization of Fibers

Fiber morphology was observed using a JEOL model JSM-6510/GS scanning electron microscope (SEM). Samples were prepared by cutting samples of the mats with a blade and fixed on aluminum stubs using double-sided adhesive tape and coated with gold using a sputtering (Balzers, SCD 050). The diameter of the fiber was measured with the aid of an image analysis software (Image J, National Institutes of Health, USA). For each experiment, the average fiber diameter and distribution were determined from approximately 100 measurements randomly taken from representative fiber morphology.

Thermal analysis was conducted by differential scanning calorimetry (DSC) in a Q100 TA Instruments. Samples were crimped in aluminum pans and heated at a rate of 10oC/min, from 0oC to 180oC in a nitrogen atmosphere.

3.RESULTS AND DISCUSSION

3.1Rheology

The viscosity data for each of the polymeric solutions in is presented in Fig. 1, revealing differences in their properties as a function of hormone concentration.

(a) (b)

Figure 1. Variation in viscosity as a function of the shear rate for PLA (a) and zein (b) solutions.

Viscosity curves for polymer solutions of neat PLA in chloroform:acetone at 3:1 (v/v) and zein in alcohol:water at 8:2 (v/v), revealed a Newtonian behavior, as well as for samples with 2 and 4% of progesterone added in PLA and 2,4 and 8% of progesterone added in zein. In general, the viscosity of the PLA and zein solutions increased as the concentrations of progesterone increases. Moreover, a pseudoplastic behavior was observed to PLA+ 8% P4 samples. The effect of the presence of progesterone in increasing PLA and zein solutions viscosity can be explained in terms of the interaction forces taking place between the progesterone and the biopolymer. Depending on the type and intensity of these interactions, rheological properties of polymer solutions can changed and thus affect the releasing behavior when in the solid state. The increase in the viscosity of PLA and zein solutions by the addition of progesterone indicates interactions between the polymer and the progesterone.

3.2Poly(lactic acid) fiber mophology

Figure 2 shows SEM images of solution blow spinning nanofibers obtained from PLA plus progesterone additions of 2; 4 and 8 %wt as collected on the rotating drum.

(a) (b)

(c) (d)

Figure 2. Poly(lactic acid) fiber morphology: (a) neat, (b) 2%P4, (c) 4% P4 and (d) 8%P4

Solution blow spun fibrous membranes with uniform morphology and a narrow size distribution were produced. The hormone-free and the hormone loaded PLA fibers have both a smooth surface and no progesterone crystals were observed on fiber surface. This suggested that progesterone was dispersed homogeneously in the solution and into the spun fibers. The average diameters ranged from 289 nm for PLA fibers to about 440 nm for fibers with progesterone (Table 3). The increase in the average fiber diameter due to active agents addition can be interpreted as a success in the incorporation of P4 in the PLA matrix.

Table 3 Solution Blow Spinning fiber diameter

Fiber

Progesterone added

(% wt)

Resulting fiber diameter

(nm)

PLA

0

289 ± 92

PLA

2

283 ± 80

PLA

4

441 ± 180

PLA

8

398 ± 220

3.3Zein fiber morphology

Figure 3 shows SEM images of electrospun nanofibers obtained from zein plus progesterone additions of 2; 4 and 8 %wt.

(a) (b)

(c) (d)

Figure 3. Zein fiber morphology: (a) neat, (b) 2%P4, (c) 4% P4 and (d) 8%P4

Neat zein fibrous membranes with uniform morphology and a narrow size distribution were produced. As PLA case, the hormone-free and the hormone loaded zein fibers have both a smooth surface and no progesterone crystals were observed on fiber surface. This suggested that progesterone was dispersed homogeneously in the solution and into the electrospun fibers. Moreover, beaded fibers were observed to zein+ hormone samples. The effect of the presence of progesterone in increasing bead concentration in zein fibers can be explained by polymer/drug/solvent interactions. Progesterone is soluble in alcohol phase but not in water phase. This fact leads changes in rheological properties of these solutions that cannot be observed in PLA+ progesterone solutions.

The average diameters ranged from 146 nm for zein fibers to about 79 nm for fibers with progesterone (Table 4). The decrease in the average fiber diameter due to active agents addition can be interpreted as increase in the bead concentration.

Table 4 Electrospun fiber diameter

Fiber

Progesterone added

(% wt)

Resulting fiber diameter

(nm)

Bead Concentration (beads/µm2)

zein

0

146 ± 33

0

zein

2

143 ± 46

0.04

zein

4

95 ± 25

0.2

zein

8

79 ± 29

1

3.4Thermal Properties

DSC was used to determine melting temperature (Tm), glass transition temperature (Tg), and the heat of fusion (ΔHf) for neat PLA and zein nanofibers and biopolymer/progesterone nanofibers as shown in tables 5 and 6.

Table 5. Characteristic temperatures and heat of fusion (ΔHf) for solution blow spinning nanofibers

Thermal Properties

PLA Fibers

Neat

2% P4

4% P4

8%P4

Tg (oC)

59

57

56

56

Tm (oC)

150

145

147

147

ΔHf (J/g)

23

20

21

21

For PLA fibers both melting (Tm) and glass transition temperature (Tg) decreases slightly as the progesterone content increases. In general, reductions observed in the melting point in a polymeric blend are normally associated to alteration on morphological effects (e.g., a decrease in lamellar thickness) and to a weak polymer-polymer interactions. In other words, the progesterone seems to have a plasticizer behavior within the PLA polymer matrix. Similarly, the change in glass transition temperature is generally explained as a consequence of polymer/plasticizer interactions such as due to dipole forces. In particular, a decrease in Tg is also associated to plasticizer effect of the additive.

Table 6. Characteristic temperatures and heat of fusion (ΔHf) for electropsun nanofibers

Thermal Properties

Zein Fibers

Neat

2% P4

4% P4

8% P4

Tg (oC)

130

137

137

138

Tm (oC)

83

79

78

74

ΔHf (J/g)

114

110

111

36

Glass Transition temperature for zein fibers was increased as the progesterone contente. Probably, this change can associated to alteration on protein structure of zein. Interactions between zein and progesterone lead to a decrease of segmental mobility of macromolecules. Water melting temperature and heat of fusion are sightly decreased by progesterone.

3.5Hormone Release

Figure 4 illustrates the release behaviors of progesterone from the nanostructured PLA membranes. It is observed that the difference between the PLA/P4 systems releasing is basically related to the total amount of progesterone released (i.e., a determined amount of PLA+8%P4 will release more progesterone in the media than the same quantity of PLA+4%P4, which, consequently, will release more progesterone in the media than the PLA+2%P4 fiber). However, the release comportment of each system is basically the same, obeying a first order kinetic of liberation with very similar K constant values. Moreover, Fig. 4 showed that the half-life for all samples is between 4 and 5 hours.

Figure 4. In Vitro release profile of progesterone from PLA membranes.

4.CONCLUSIONS

The results of the current study confirm some miscibility of progesterone and hydrophobic biopolymers according to rheology, and thermal used methods. Progesterone was found to have the effect as plasticizer when added to PLA nanofibers. The experimental data display a decrease in the Tg values, which is generally attributed to polymer/active agent interactions. Thus, nanofiber technology as electrospinning and solution blow spinning can be appropriated used to encapsulate active agents in biodegradable and biocompatible polymers, providing a release with first-order kinetics rates. This study clearly indicated that PLA and zein nanofibers can be potentially used in controlled delivering system of progesterone, being a powerfull to in the control of the estrus cycle in livestock animals.

ACKNOWLEDGEMENTS

The authors are grateful for the financial support from FINEP/MCT, CAPES, CNPq, Embrapa/LNNA/Rede AgroNano, and FAPESP (Process No 2010/19860-1) funding agencies.

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0

20

40

60

80

100

28

32

36

40

Viscosity (mPa.s)

Shear rate (s

-1

)

zein

zein+2% P4

zein+4% P4

zein+8% P4

1 μm